Chemical attachment of hydrophilic polyalkylene glycol linear polymers (known as PAG) to biopharmaceuticals such as proteins and peptides is well known and commonly used in biotechnology. The most common polyalkylene glycol molecule is polyethylene glycol polymer (known as PEG).
As an example of biotechnical application of PAG, some active derivatives of PAG have been attached to biopharmaceuticals such as proteins and enzymes with beneficial results. Since PAG is soluble in organic solvents, PAG attached to such biopharmaceuticals as enzymes or proteins can produce resulting conjugates that are soluble and active in organic solvents. Attachment of PAG to protein can reduce the immunogenicity and rate of kidney clearance of the PAG-protein conjugate as compared to the unmodified protein. In addition attachment of PAG to biopharmaceuticals such as protein can also dramatically increase the blood circulation lifetimes for these PAG conjugates.
In preparing PAG conjugates with biopharmaceuticals such as proteins the pharmacokinetics of the particular biopharmaceuticals will govern both the efficacy and duration of effect of the drug. In view of the immunogenicity, water insolubility and short in vivo half-life of biopharmaceuticals particularly proteins and polypeptides, it has become of major importance to reduce the rate of clearance of these biopharmaceuticals so that prolonged action can be achieved. This may be accomplished by retarding or inhibiting glomerular filtration which can be effected both by the charge on the protein and its molecular size (Brenner et al., (1978) Am. J. Physiol., 234, F455). By increasing the molecular volume and by masking potential epitope sites, modification of a therapeutic biopharmaceutical such as a polypeptide and a protein with a polymer has been shown to be efficacious in reducing both the rate of clearance as well as the antigenicity of the biopharmaceutical, especially proteins. Reduced proteolysis, increased water solubility, reduced renal clearance, and steric hindrance to receptor-mediated clearance are a number of mechanisms by which the attachment of a PAG polymer to the backbone of biopharmaceuticals such as polypeptides and proteins are beneficial in enhancing the pharmacokinetic properties of the drug Thus Davis et al., U.S. Pat. No. 4,129,337 discloses conjugating PEG to proteins such as enzymes and insulin to produce a less immunogenic product while retaining a substantial proportion of the biological activity of these biopharmaceuticals.
There are a large variety of active PAGs, particularly PEG, reagents which have been developed for the modification of biopharmaceuticals such as proteins (see for example Zalipsky, et al., and Harris et al. in: Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21 and 22), most of which require the formation of a linking group between PEG and the biopharmaceuticals. Some of these reagents are unstable, to various degrees, in the aqueous medium in which the conjugation reaction occurs. In addition the conjugation process often results in the loss of in vitro biological activity which is due to several factors foremost of which being a steric interference with the proteins active site. A desired property therefore of a new reagent would be one that is not susceptible to degradation in an aqueous medium and one which may be employed to produce the site specific modification of a protein.